Method for tissue expansion using pulsatile motion

ABSTRACT

A method of increasing the rate of expansion of tissue area and volume, either in vivo or in vitro, which comprises preparing a tissue for expansion and subjecting the tissue to stretching forces, wherein the stretching forces are alternatively increased and decreased to provide alternating periods of stretch and relax cycles.

TECHNICAL FIELD

[0001] This invention resides jointly in the fields of surgery andtissue culture and is particularly directed to the expansion of animaltissue from an original, natural size to a larger size, usually with theintention of using the expanded tissue to replace defective tissue inthe body of a living patient.

BACKGROUND

[0002] Tissue replacement is an essential component of reconstructivesurgery after burns, trauma, tumor excision, and correction ofcongenital anomalies. For example, there are approximately 1 millionburns per year in the U.S. alone, which result in about 100,000admissions to burn units, about ⅓ of which require skin grafting.

[0003] The best possible skin available for grafting would be skin fromthe same patient taken from a donor site elsewhere on the body (referredto as an autograft). Suitable skin graft donor sites, however, arelimited not only by body surface area, but can also be affected byprevious graft harvest or trauma. There are times, when donor skin islimited and the amount of skin required for grafting is quite large,that sufficient autografts are not available. Because of the importanceof the skin in preventing infection, either the donor skin must be usedto cover a larger area than it originally covered or some suitablereplacement material must be used. Harvesting of multiple skin graftsfrom the same donor site is often used, but such harvesting requiresweeks to months between procedures for new skin to grow on the donorsite. It is also a very traumatic technique, since multiple painfuloperations must be undertaken.

[0004] In a similar manner, other tissues also require replacement aftertraumatic injury, tumor excision, and other medical situations involvingtissue loss. Autografts are preferred for muscle, cartilage, tendon,nerve, and other tissue replacement whenever possible in order to reducehost vs. graft immunity issues. Under appropriate circumstances, donortissues derived from sources other than the recipient are acceptable forboth skin and other tissues, but usually only as temporary replacements.For example, in patients suffering from large burns with limited donorskin sites, cadaver allografts are commonly used for temporary skincoverage, but ultimately such allografts are rejected and a permanentautograft is required. In addition, allografts also pose a risk ofinfection of the recipient by viruses or other disease-causing organismspresent in the donor, such as infection by human immunodeficiency virusor hepatitis virus.

[0005] Artificial tissues have been developed in order to avoid theproblems associated with allografts. For example, to aid in the graftingof skin on patients with limited donor areas, cultured epithelial cellsderived from the patient being treated have been utilized in manygrafting applications. In general, the cells are used in the form of amonolayer of epithelial cells grown on a culture medium. Preparation ofsuch cultures requires many weeks or months, and the product is quitedifficult to handle because of its fragility, even when multipleepidermal cell layers are used to form a multi-layer skin substitute.

[0006] Tissue expansion techniques, which were developed as in vivotechniques, have been used in plastic surgery for over a decade and canbe helpful in increasing the area of donor tissue. Skin is not the onlytissue that can be and has been expanded, although it is the mostcommon. Other tissues have been expanded in surgical and other in vivosituations. Arteries, peripheral nerves, and skin have all been expandedin human clinical trials. Ureter, small bowel, and bladder have beenexpanded in animal trials.

[0007] The techniques used for in vivo tissue expansion are similar forall tissue types and involve mechanically stretching the tissue whilethe tissue is still attached to the patient's body. For example, byplacing an expander subcutaneously and injecting it with saline, skincan be expanded and its surface area increased. This allowsreconstruction with local skin after expansion of an adjacent tissuebed.

[0008] Background information in the general field of tissue expansion,including techniques suitable for skin grafting and tissue replacement,is available in the patent and scientific literature. A number ofexemplary patents and scientific publications are cited below, both asexamples of existing technology and to provide additional basis andsupport for ancillary technology related to the practice of the presentinvention:

[0009] U.S. Pat. No. 5,882,353 entitled “Mechanical tissue expander”

[0010] U.S. Pat. No. 5,858,003 entitled “Systems and methods forpromoting tissue growth”

[0011] U.S. Pat. No. 5,855,588 entitled “Combination dissector andexpander”

[0012] U.S. Pat. No. 5,788,627 entitled “Cavernosal extension implants”

[0013] U.S. Pat. No. 5,776,159 entitled “Combination dissector andexpander”

[0014] U.S. Pat. No. 5,630,843 entitled “Double chamber tissue expander”

[0015] U.S. Pat. No. 5,618,310 entitled “Tissue, expansion andapproximation device”

[0016] U.S. Pat. No. 5,549,713 entitled “Method for skin tissueexpansion”

[0017] U.S. Pat. No. 5,507,775 entitled “Tissue expansion andapproximation device”

[0018] U.S. Pat. No. 5,476,479 entitled “Handle for endoscopic surgicalinstruments and jaw structure”

[0019] U.S. Pat. No. 5,441,540 entitled “Method and apparatus for skintissue expansion”

[0020] U.S. Pat. No. 5,425,760 entitled “Tissue expander apparatus, andmethods of constructing and utilizing same”

[0021] U.S. Pat. No. 5,158,571 entitled “Tissue expander and method forexpanding tissue”

[0022] U.S. Pat. No. 5,092,348 entitled “Textured tissue expander”

[0023] U.S. Pat. No. 5,005,591 entitled “Self-inflating tissue expander”

[0024] U.S. Pat. No. 4,904,267 entitled “Method and device for fixing ajoint prosthesis”

[0025] U.S. Pat. No. 4,863,469 entitled “Method and apparatus forexpanding nerve tissue”

[0026] U.S. Pat. No. 4,828,560 entitled “Spring ring tissue expander”

[0027] U.S. Pat. No. 4,800,901 entitled “Balloon-type Tissue expansiondevice”

[0028] U.S. Pat. No. 4,643,733 entitled “Permanent reconstructionimplant and method of performing human tissue expansion”

[0029] U.S. Pat. No. 4,157,085 entitled “Surgically implantable tissueexpanding device and the method of its use”

[0030] Argenta, “Controlled tissue expansion in reconstructive tissue,”Brit. J. Plas. Surg., 37:520-529 (1984)

[0031] Argenta et al., “The Use of Tissue Expansion in Head and NeckReconstruction,” Ann. Plast. Surg., 11:31-37 (1983).

[0032] Arons et al., “The surgical applications and implications ofcultured human epidermis: A comprehensive review,” Surgery, 111:4-11(1992)

[0033] Carney, “Generation of autograft; the state of the art,” Burns,12:231-235 (1986).

[0034] Chen, “An animal experiment on short gut lengthening,” Chin. Med.J. (Engl.), 110:354-357 (1997).

[0035] Gallico, “Biologic Skin Substitutes,” Clinics in Plastic Surgery,17:519-526 (1990)

[0036] Greenwald et al., “Full-Thickness Skin Wound Explants in TissueCultures: A Mechanical Evaluation of Healing,” Plastic andReconstructive Surgery, 90:289-294 (1992)

[0037] Kirsner et al, “The Biology of Skin Grafts,” Arch. Dermatol.,129:481-483 (1993)

[0038] Liatsikos et al, “Tissue expansion: a promising trend forreconstruction in urology,” J. Endourol., 14:93-96 (2000).

[0039] Nanchahal and Ward, “New grafts for old? A review of alternativesto autologous skin,” Brit. J. Plas. Surg., 45:354-363 (1992)

[0040] Satar and Atala, “Progressive dilation for bladder tissueexpansion,” J. Urol., 162:829-831 (1999).

[0041] Stifelman and Hensle, “Ureteral tissue expansion for bladderaugmentation: a long term prospective controlled trial in a porcinemodel,” J. Urol., 160:1826-1829 (1998).

[0042] Sung Shin Wee et al., “Continuous versus intraoperationalexpansion in the pig model,” Plastic and Reconstructive Surgery,90:808-814 (1992)

[0043] A particularly useful advance in the field of tissue expansionwas initiated by Dr. Joshua Korman, who developed the first process forin vitro skin expansion in the 1990s. The investigations of Dr. Kormanresulted in the issuance of two U.S. patents, U.S. Pat. No. 5,686,303,entitled “Method of Growing Vertebrate Skin In Vitro,” and U.S. Pat. No.5,914,264, entitled “Apparatus for Growing Vertebrate Skin In Vitro.”The method involves growing complete vertebrate skin in vitro byobtaining a segment of vertebrate skin, positioning the skin segment inan artificial cell-growth medium containing sufficient nutrients tomaintain growth of cells of the skin, and subjecting the skin segment tostretching forces while the skin segment is in the medium. Skin producedby the method and an apparatus for carrying out the method were alsodisclosed in these patents.

[0044] Even this improvement, which eliminates much of the pain anddiscomfort associated with in vivo skin expansion, can itself beimproved by increasing the rate of tissue expansion in order to improvethe life of the patient who is waiting for tissue expansion to becompleted so that a defective tissue can be replaced with the expandedtissue.

[0045] In the past, investigations in tissue expansion have demonstratedthat use of continuous expansion forces in post operative situationsover a period of three days or more is superior to intraoperative tissueexpansion, even when the intraoperative procedure involved threethree-minute cycles of pressure increase and decrease. See, Sung ShinWee et al., “Continuous versus intraoperative expansion in the pigmodel,” Plastic and Reconstructive Surgery, 90:5, 808-814 (1992).Although “cycling of pressure” is mentioned (along with other factors;p.811) in a section discussing potential additional skin expansion,there is no indication that the rate of expansion would be increased bysuch cycling. Instead, there is an indication that the total volume ofexpansion did not plateau in the study, so that a greater total volumemight be obtained by use of the techniques mentioned, which includehigher pressures, cycling of pressure, longer time periods, and ananimal that did not lie on the expander and interrupt the expansionprocess.

[0046] Accordingly, it remains desirable to develop a technique thatwill improve the rate of tissue expansion. Investigations on rateimprovement have continued in the laboratory founded by Dr. Korman, andresults of those investigations are the subject of the present patentapplication.

SUMMARY OF THE INVENTION

[0047] Accordingly, it is an object of the invention to increase therate of tissue expansion in all types of expansion, whether in vivo orin vitro. Although skin is the tissue most often expanded, it is anobject of the invention described herein to apply the method to alltypes of living tissue.

[0048] It is a particular object of the invention to provide a tissueautograft having a larger volume and/or surface area than that of thedonor graft site in as rapid a manner as possible.

[0049] These and other objects of the invention have been accomplishedby providing a method of increasing the rate of expansion of tissue areaand volume, either in vivo or in vitro. The method comprises preparing atissue for expansion, the tissue having an initial area and volume, andsubjecting the tissue to stretching forces that are alternativelyincreased and decreased in a controlled manner, rather than beingmaintained at a steady state. The increases and decreases in stretchingforces are referred to as being positively modified in order todistinguish them from naturally occurring changes that may occur, forexample, when movement of the body (in an in vivo expansion) causes anincrease in the pressure exerted by a subcutaneous fluid expander orwhen stretching forces decrease naturally as tissue growth occurs (foreither in vivo or in vitro expansion). Although the positivelycontrolled increase and decrease in stretching forces can occur at anyof a variety of cycle rates, manners, and durations (e.g., sinusoidalincrease and decrease or rapid expansion followed by rapid forcereduction), minimally there is at least one cycle per day for a periodof at least one day. The expanded tissue is available for use inreconstructive surgery (or any other purpose) as soon as it has expandedto the desired size. Experimental evidence has shown that the growth intissue size is more rapid relative to similar methods that operatewithout alternatively and positively increasing and decreasing thestretching forces.

[0050] In various embodiments of the invention, the stretching forcesare orthogonal or radial relative to each other, but they can also be ofdifferent geometry, such as linear stretching of elongated tissues suchas ligament or nerve tissue. The tissue is preferably human for use inhuman recipients, but veterinary use of the new skin is also encompassedby the invention. Other embodiments of the invention are set forth belowin detail.

DESCRIPTION OF SPECIFIC EMBODIMENTS

[0051] The present invention combines a number of previously knowntechniques in a novel manner to achieve results not previously obtained.Namely, by alternatively and positively increasing and decreasing thestretching forces applied to tissue, either in vivo while the tissue isattached to a patient (or other living creature) or in vitro duringtissue culture, size of the tissue can be increased more rapidly thanwas previously achieved using constant pressure. The reason for thisincreased growth rate is not known, but it is believed to be at least inpart the result of providing a rest phase for tissue growth after thestress of a stretching force that realigns and/or breaks down collagenand/or other structural components of tissues. Potentially thistechnique could save costs by minimizing the number of surgicalprocedures a patient would require while also decreasing the riskassociated with multiple anesthetics and surgical procedures sometimesneeded in cases of complex reconstruction. Patient suffering will alsobe reduced, as there will be a decrease in the time that a patient mustwait for tissue expansion to be completed.

[0052] There are a number of technologies ancillary to the presentinvention that are well developed and that thus will not be describedhere in great detail, such as methods for excision of the original skinor other tissue segment, production of culture media for the growth andmaintenance of intact tissue when an in vitro method is used, andgrafting techniques for the attachment of the graft to the host. Suchmethods and materials are exemplified here and references are given toscientific publications, where appropriate, so that the invention canreadily be practiced. It will be recognized, however, by one of ordinaryskill in the art, that many variations of these ancillary methods andmaterials exist and that the invention is not limited to the specificexamples provided here.

[0053] In general, the method of the invention provides a method ofenlarging tissue taken from a donor site of an animal donor (whichincludes humans). There is no limit on the tissue, other than that it isliving tissue. Examples include skin, cartilage, tendon, ligament, bloodvessel, nerve, and bone for use in in vitro expansions. The same tissuesare preferred in in vivo applications, along with ureter and small boweltissues. Other tissues can be expanded, but are less preferred.

[0054] Often the donor tissue is being expanded for the purpose ofproviding an autograft capable of covering both the original donor site,as well as a different site where tissue replacement is desired.However, the enlarged tissue is also useful for replacement of adifferent tissue without replacement of the original donor site (such asoccurs with the harvesting of a tendon from one location of a body withthe intent of replacing a different, more important tendon). Decisionson suitable donor sites are usually made by a physician and will vary,depending on the injury or disease site being repaired. Examples ofdonor sites and tissue collection are given in the references sitedabove that describe prior techniques for tissue expansion carried outprior to the present invention.

[0055] In the following description of various embodiments of theinvention, the invention is described in most cases as being practicedwith a human autograft. However, the invention is not so limited and canbe used for both allografts (within the same species, but with the donorand recipient being different individuals) and xenografts (donor andrecipient from different species). Additionally, the invention is notlimited to preparation of tissue obtained from human donors, since itcan be advantageously practiced to produce tissue of variousvertebrates, either for veterinary use as autografts or allografts, orfor use in the production of xenografts in humans (which would normallyrequire suppression of the immune system of the human recipient when theproduct tissue is used as a graft). In a similar manner, artificial (butliving) human or non-human tissue can be used as donor tissue for tissueenlargement. Such artificial tissues are typically those produced bygenetic manipulation of natural tissues, such as by incorporation of agene into cells of a donor tissue. Such artificial tissues are typicallythose produced by isolation of cells from donor tissue that are thenincorporated into an artificial or modified biological substrate tocreate a tissue or genetic modification of a natural tissue, such as byincorporation of a gene into cells of a donor tissue.

[0056] A segment of vertebrate tissue (graft donor segment) is obtainedby any of the techniques normally available for this purpose, usuallysurgical excision (for full-thickness skin and most other tissues) oruse of a dermatome (for split-thickness skin). If a dermatome (i.e., anyplane-like device for removing skin from a subject) is used, thethickness of the layer should be selected to ensure that at least someof the dermal layer is present. The tissue size will vary depending onthe use for which it is intended and will vary from species to speciesand even from location to location on the body of an individual. Forexample, a typical setting for a dermatome used to preparesplit-thickness human skin is about {fraction (12/1000)}th of an inch(about 0.3 mm).

[0057] Full-thickness skin segments for use in in vitro techniques aregenerally obtained by surgical excision, while split-layer skin segmentsfor in vitro techniques are obtained by a dermatome. Both of thesetechniques, as well as other general techniques in the field of skingrafting, as described in Chapter 1 (pp. 1-90) of Grabb and Smith,Plastic Surgery, Little Brown & Company, Boston, Mass., USA, 4th Ed.(1991), James W. Smith and Sherrel J. Aston, eds. For tissue types otherthan skin intended for in vitro expansion, the donor tissue is obtainedby surgical excision.

[0058] When intended for in vitro tissue expansion, the detached skin(or other tissue) is normally transferred directly to a culture mediumand in most cases is not allowed to dry out before being positioned inthe medium. The shape of the detached tissue segment is not material tothe practice of the invention, but is often selected depending on theintended final use. Additionally, certain shapes will be better suitedto individual specific apparatus variations described here. Selectionand manipulation of skin or other tissue used with an in vivo expanderwill be made by a physician using standard techniques in the field ofreconstructive surgery, as is described in the numerous referencespreviously cited.

[0059] The size of the donor tissue is generally selected for theconvenience of use, taking into consideration the intended final use,the amount of tissue available for use as donor tissue, and theapparatus that will be used to supply the stretching forces. Typicalhuman skin segments are from 1 by 1 cm to 10 by 30 cm but can varysignificantly depending on the availability of donor skin. There isgenerally no impact of graft size on the method of the invention, sothat surgical and other procedures generally are more important indetermining tissue size. For ease of handling in surgical skin grafting,segments ranging in size from 5 by 5 cm to 15 by 15 cm are preferred forin vitro uses. Similar dimensions are typical for other tissues.

[0060] Artificial cell-growth media containing sufficient nutrients tomaintain growth of cells of a tissue segment are well established andneed not be described here in detail for the in vitro techniques. Suchmedia are also referred to as nutrient media or tissue-culture media.Whether any given medium will be satisfactory (if not already known) caneasily be determined experimentally using the procedures for tissuegrowth set out in the examples below. Many such media are commerciallyavailable, such as Dulbecco's modified Eagle's medium (DMEM) with 10%added fetal calf serum. Other suitable media include basal medium(Eagle) with Hanks's BSS (85%) supplemented with calf serum (15%) andHam's F12 mediun (90%) supplemented with fetal bovine serum (10%). Whenserum is used to supplement an artificial medium, fetal serum ispreferred, especially fetal serum from the same species as the recipientof the graft. When this is not possible or ethically desirable, therecipient's own serum can be used. For a number of media that can beused to maintain and grow tissue, see, for example, the mediaformulations section of any volume of the American Type CultureCollection publication entitled Catalogue of Cell Lines & Hybridomas(e.g., 5th edition, 1985, pages 265-273). This ATCC publication alsocontains information (in connection with specific tissue-derived celllines) on which media are best for use with tissue or cell culturesderived from a specific tissue.

[0061] The present invention is described herein as a method. Variousembodiments of an apparatus in which the method can be practiced aredescribed below. However, a second application from the laboratory ofthe present inventions has been concurrently filed with the presentmethods application and is directed to apparatus for use with thismethod. The apparatus application names common inventors, is filed onthe same day, is entitled “Apparatus for Tissue Expansion usingPulsatile Motion,” and is identified by attorney docket numberRECT003/00US. As discussed in that application (and summarized below),there are no specific limits that the method places on the apparatus, sothat the method can be carried out in a variety of apparatuses (or evenmanually, although that is less preferred because of the obviousinconvenience of manual operation). Although various embodiments of anapparatus designed to be used with the present invention will haveindividual advantages, usually related to ease of use, preferred aspectsof the method can be practiced with any apparatus.

[0062] The apparatus in which in vitro tissue culture and stretchingtakes place can vary widely, being either simple or complex. An exampleof a simple apparatus is a Petri (or similar) dish containing atissue-culture medium and having a set of clamps, wires, pulleys, andweights arranged so that the clamps can be attached to a tissue segmentin the medium and subjected to alternatively increased and decreasedforces applied to clamps by weights attached to the clamps by the wiresand suspended by the pulleys to reduce friction. The weights can then bechanged on a schedule to provide the required variation in force. A morecomplex apparatus could contain electric motors for supplyingalternating levels of force to the clamps or for circulating the culturemedium in the apparatus, sensors to measure forces and stretchingdistances, reservoirs for fresh and waste medium, controlledatmospheres, and the like. An apparatus intended for in vitro use withthe present method will comprise a container for holding a tissueculture medium, at least two connectors for holding a detached tissuesegment in the culture medium, and a component that supplies opposingforces via the tissue connectors to the tissue segment (often referredto as the power sub-component). The container can be an integral part ofthe apparatus or can be a separate container that is retained by theapparatus at a specific location. In the later case the connectors are“in” the container by being affixed to the apparatus that will engagethe container.

[0063] An in vitro alternative apparatus can comprise a tubular fluidreservoir having an open end, a clamp located at the open end of thereservoir, where the clamp is adapted to seal the skin segment over theopen end to provide a fluid-tight seal, and means for supplyinghydrostatic pressure to a fluid located in the reservoir. It should berecognized here that “tubular” does not require a circular crosssection, as the word is used here. Examples of means for supplyinghydrostatic pressure comprises (1) a nutrient reservoir fluidlyconnected to the tubular fluid reservoir and being located at a highergravitational potential than the skin segment when the apparatus islocated in its normal operating position or (2) a pump fluidly connectedto the tubular reservoir. A pump is any mechanical device that movesfluid from one location to another and includes a hydraulic piston. Anexample of a suitable clamp would be an annular member adapted to fittightly against a flange on the open end of the tubular reservoir, withholes or grooves in the flange or annular member (or both) adapted tocontain screws, bolts, wing nuts, or the like for fastening the annularmember against the flange, with the skin segment being located betweenthem to provide a fluid-tight seal at the end of the tubular reservoirwith the skin being attached over the end of the reservoir as in a drum.Since freshly harvested skin is resilient (as are many other planartissues, such as mucosal linings), no additional seal is required, but aflexible sealing member (such as an O-ring) can be provided between,e.g., the flange and annular member, if desired. Changes in the desiredforces can be provided by moving the reservoir up and down relative tothe tissue or by changing the pressure supplied by the pump.

[0064] In a similar manner, both simple and complex in vivo tissueexpanders can be adapted to the method of the present invention. Suchapparatuses typically comprise an expandable balloon-like structure thatis implanted subcutaneously in the patent. Access ports are provided sothat fluid can be injected into the balloon, causing it to expand andexert the stretching forces. Simple devices consist entirely of theballoon-like expander and a septum through which fluid can be manuallyinjected through the skin via a syringe. More complex devices havepercutaneous catheters leading to external pumps and pressurecontrollers, specific geometries intended for expansion of differenttissue (or different locations of the same tissue, e.g., different skinareas), and other apparatus components.

[0065] One example of these more complex apparatuses that includepressure controllers is provided in U.S. Pat. No. 4,955,905. This patentdiscloses a pressure monitor for use in connection with tissue expanderenvelopes implanted beneath the tissue of the skin wherein a liquid isinjected into the envelope to cause expansion of the skin or tissue andadditional liquid is periodically injected to cause progressivelyincreased expansion of the tissue. The tissue monitor includes meansestablishing direct communication between the pressure monitor and theliquid injected under pressure whereby the monitor will provide areading of the internal liquid pressure in the envelope. The methoddescribed in this patent comprises the steps of implanting an inflatableenvelope beneath the skin, injecting a sterile solution under pressurethrough a fill line communicating with the envelope, interrupting theflow of liquid under pressure into the envelope, sensing the pressurelevel of liquid injected into the envelope, and adjusting the pressurelevel when necessary either by removing from or injecting liquid intothe envelope.

[0066] This last operation superficially resembles the method of thepresent invention, but differs in that there is no positive cycling ofpressure, simply a monitoring of pressure, followed by an immediateadjustment if the envelope has been filled with too much liquid. This isindicated by a description in the specification that describes theevents that occur in a filling sequence. After each filling sequence,the internal pressure of the envelope is read from the monitor (theinternal pressure corresponds to the pressure exerted upon thesurrounding tissue). If the pressure is less than desired, the fillingsequence can be repeated. If the pressure is too high and thought tohave the potential to cause tissue necrosis, saline solution may beremoved directly from the envelope until the desired pressure isattained.

[0067] A specialized area of in vivo tissue expansion, particularly forskin, involves wound closing. Wounds that are large and open, thatresult from acute tissue loss, or that are infected do not lendthemselves to traditional tissue expansion due to the lengthy expansiontime previously required. A number of devices and techniques haveemerged that are intended to take advantage of the elastic nature ofskin by pulling the wound closed without irreparably damaging the skinadjacent to the open wound. These methods use attachment to the skin onopposing sides of the wound through the use of hooks, needles, sutures,or adhesives and then apply either adjustable or constant tension on theopposing wound edges to slowly close the would. Some devices requiremanual re-tensioning as the wound closes, while others maintain aconstant tension over time through some form of automated re-tensioning.This process can take up to three days, depending on the size of theopen wound. The present invention of tissue expansion using pulsatilemotion can be directly applied to this specific area of tissueexpansion, by applying alternating periods of stretching and relaxing asdescribed herein, rather than the constant tensions used in the past.

[0068] A number of static wound-closure devices have been described inthe prior art, including U.S. Pat. Nos. 5,649,960 (“Apparatus and Methodfor Accelerating the Stretching of Skin”), 5,759,193 (“Single NeedleSkin Stretching Device”), 4,526,173 (“Skin Closure Device”), 5,486,196and 5,893,879 (both entitled “Apparatus for the Closure of Wide SkinDefects by Stretching of Skin”), 5,234,462 (“Method and Kit forAccelerating the Closing of Open Skin Wounds”), 5,549,713 (“Method forSkin Tissue Expansion”), 4,825,866 (“Wound Closure Device”), and5,127,412 (“Skin Tensioning”). Also see chapter 25, entitled“Alternative Devices and Techniques” in Tissue Expansion, Ralf E. ANordstram, ed., Butterworth-Heinemann (1996). These devices, althoughused in in vivo situations, differ from balloon-like tissue expanders,in that that are attached externally to skin rather than being implantedinternally in a patients body. Any of these devices can be used with themethod of the present invention, either by manual operation of thedevices so as to provide a series of cycles that add and remove tensionon the skin or by modifying the devices so that such cycles are producedautomatically.

[0069] A key aspect of the present invention is subjecting a tissue tostretching forces that are positively controlled so that the tissueexperiences a “stretch and relax” cycle. Here “stretching forces” meansa force or forces applied to the tissue in one or more direction inwhich expansion (i.e., tissue growth) is desired. Because of thephysical nature of stretching forces, at least two opposed forces areapplied to a tissue (because of Newton's familiar law of equal andopposite forces). If only two opposed forces are present the stretchingis along a line coaxial with the stretching forces, subject to someadditional stretching along adjacent regions of the tissue. This is thesimplest stretching situation, but is not particularly desired (otherthan for substantially linear tissues, such as nerve, tendon, andligament tissue) because of the resulting tissue deformation. Additionalforces can be applied to provide for more regular stretching ofvolumetric or planar tissues, such as muscle or skin. Parallel opposedforces (such as would be applied by two broad, rigid clamps attached toopposite ends of a detached skin segment) lead to stretching along asingle dimension of a tissue. Non-parallel multiple stretching forces(e.g., radial outward from a central point or orthogonally in the planeof a tissue) result in stretching in both of the two dimensions of aplanar tissue (e.g., the two dimensions parallel to the plane of a skinsurface). Forces can be supplied in three dimensions as well.

[0070] Forces can be applied to planar tissues, such as skin, that arenot entirely parallel to the skin surface. However, some portion of theforce must be parallel to the planar surface for stretching to takeplace. For example, when used with in vitro stretching of skin, a convexsolid surface or a fluid forced against the face of a detached skinsegment whose edges are fixed will cause the skin segment to besubjected to forces both orthogonal and parallel to the surface of theskin; such stretching comes within the scope of the present invention.This is the type of stretching of skin that occurs with thefluid-expandable balloons and envelopes that are common in in vivo skinexpansion.

[0071] When a detached tissue segment is being stretched in an in vitroapplication, the ends of the segment are held in place in the tissueculture by some physical apparatus. Any apparatus that can be used tohold the ends in place can be used. An attachment apparatus is neededfor each point to which a force will be applied. Typical attachmentapparatuses include clamps, hooks, sutures, and glue. A clamp can benarrow (e.g., less than {fraction (1/10)} the length of the edge beingheld) or broad (up to or greater than the width of the edge, andgenerally considered broad when greater in width than ½ the width of theedge of the tissue being clamped). If opposed broad clamps are used,stretching between the ends of the clamp will generally be restricted ifan orthogonal stretching force is also present on the skin. For maximumstretching efficiency, multiple attachment points capable of moving awayfrom each other during the stretching process are preferred. Forexample, multiple small hooks or clamps attached in a generally circularmanner to a circular detached skin segment and subjected to forcesapplied radially outward from the center of the segment automaticallymove away from each other as stretching proceeds, thus supplyingstretching forces along the tangents of the circle as well as along itsradii.

[0072] The forces themselves can be supplied by any means for supplyingforce, such as a weight, spring, or motor. The force being applied atany given point of the cycle can be either static or dynamic. Here astatic force is one that is applied between two attachment points thatdo not move further apart from each other as cell growth and divisionoccurs. Such growth reduces over time the force between the attachmentpoints. For example, two clamps can be attached to opposite ends of adetached skin segment, with one (or both) of the clamps being attachedto a screw that the distance between the clamps can be varied. Turningthe screw to move the clamped ends away from each other produces aninitial force on the skin segment, but this force decreases as cells inthe skin grow and divide. A dynamic force, on the other hand, is oneprovided between two attachment points that are capable of relativemovement so that a constant force can be maintained. For example, twoclamps can be attached to weights that are suspended via a pulley systemfrom opposite ends of a detached skin segment. The force on the skinsegment in such an apparatus remains constant as the skin grows anddivides.

[0073] The amount of force applied at any given point in time to atissue during a stretch phase is minimally that required to cause thetissue to stretch and will not exceed the amount that causes the tissueto rupture. Since the strength of different tissues obtained vary (eventissues obtained from different locations of the same donor/recipient ofan autograft), the forces are best determined empirically. Minimumstretch-cycle force is that which causes some stretch to take place.Maximum stretch-cycle force is that which causes tissue necrosis.Preferably, maximum force is less (typically at least 5% less) than thatwhich causes blanching of tissue as a result of the inability of thetissue to maintain blood flow through internal blood vessels. In in vivosituations, patient pain will also be significant in determining maximumforces that can be used.

[0074] For example, with the use of human skin, stretching of at least2% per day is desired, preferably at least 5%, more preferably at least10%. Non-human skin can be either tougher or less tough (here “tough”refers to resistance to stretching) than human skin and thus may bestretched correspondingly less or more than these amounts. In generalskin can be stretched until rupture or cell death induced by the tensionof stretching, which can readily be followed by histologicalexamination. In some cases it may be desirable to keep stretching under15% per day to avoid cell death, in other cases under 12%. However, themaximum sustainable stretch rate is best determined empirically, usingthese numbers as initial guidelines. When skin is initially placed inthe nutrient medium, it should be stretched back to original in vivosize before actual stretching is measured, since skin removed from abody generally shrinks to less than its original dimensions.

[0075] In many cases, the actual forces will never be measured or known,such as in a screw-based apparatus. However, typical forces for skinrange from zero to 300 g per attachment point, preferable zero to 150 gper attachment point, using one attachment point per cm of skinperimeter. As there is variation from patient to patient in strength ofskin and other tissues, one should start with a force at the lower endof the range and increase forces gradually during the initial stretch,paying attention to counter indications, such as cracking or tearing ofthe tissue. Instead of calculating on a per attachment basis, force canbe measured per unit area. This is particularly useful for skin, as areaof coverage is often the matter of primary interest. For example, skinin the form of a disk 2.93 cm in diameter, having an area of 6.75 cm²,preferably has an applied force of 75-225 g/cm², more preferably 150g/cm². On a per attachment scale, this translates to a range of 50-200g/attachment, preferably 100 g/attachment.

[0076] Positive control of the stretch and relax cycles is an importantpart of the present invention. The “stretch” phase of a cycle occurswhile stretching force s are increasing or are being maintained at alevel that causes stretching to occur. The “relax” phase of the cycleoccurs when stretching forces are decreasing or are being maintained ata level less than the maximum used during the preceding “stretch” phase.Stretching forces on the tissue can fall to zero during the relax phase,but also can be maintained at a fraction of the force applied during thestretch phase, such as one-half, one-third, or one-quarter of themaximum stretch force.

[0077] Because a goal of the present invention is to maximize the rateof tissue expansion, the stretch phase will typically apply the maximumstretching forces that can be sustained without harm to the tissue forthe stretch period being used. Relatively higher forces have been foundto be acceptable for tissue when applied for shorter periods, whilelonger stretch cycles are expected to have better success when less thanthe maximum stretching force is applied.

[0078] Forces during the relax phase may not be zero but can bemaintained at some positive value in order to maintain some minimalstretching force on the tissue as it recovers from the stretch phase.Typically, the relax phase will have a minimum force that falls in therange of from zero to 0.9 (preferably zero to 0.5, more preferably zeroto 0.3, even more preferably zero to 0.1) times that of the maximumstretch-phase force. Most preferably the relax phase minimum will occurwith no force being applied to the tissue being expanded.

[0079] In order for the present invention to be practiced, stretch andrelax periods must alternate. The precise manner in which theyalternate, however, is less important. For example, an automatedapparatus (used either in vivo or in vitro) can cycle through a seriesof stretch and relax periods in a simple “on/off” mode (i.e., a graph ofpressure versus time would appear as a square wave). Force can alsoincrease (and later decrease) through a series of steps separated byperiods of constant force, rather than through a single increase inforce. On the other hand, use of two expandable reservoirs, with fluidbeing pumped back and forth between the two, would produce a sinusoidalgraph of stretching forces at the two locations (with each locationbeing out of phase relative to the other; i.e., one location would be ina rest phase while the other is in a stretch phase). Other techniques(such as raising and lowering of a fluid reservoir connected to a singleexpander) will also produces sinusoidal force/time effects.

[0080] Successive cycles can have the same maximum and minimum pressuresif desired, although this is not required. Because tissue expansionoccurs slowly over time, a typical pattern of pressure cycles willresult in slightly lower pressures being exerted with each cycle asstretching occurs and relieves the pressure, unless the expander isadjusted for the new resistance of the skin or other tissue beingexpanded. For example, in the on/off and two-reservoir systems justmentioned, each cycle will produce slightly lower pressure at theexpansion sites unless additional fluid is added to the system (orgreater motion of a stretching arm occurs) to make up for the volumeadded from tissue expansion. However, one can use dynamic forces or canuse force applicators (e.g., motors) with feedback control in order toprovide the same force (or a pattern of increase and/or decrease inmaximum and/or minimum force) in successive cycles, if desired.

[0081] The time period of one stretch/relax cycle can vary considerably.One cycle per day (approximately) is the minimum needed to see anincrease in expansion rate relative to static application of stretchingforces or relative to use of an initial static stretching force orpressure followed by a slow decrease in forces as tissue expansionoccurs. Rapid cycling is also possible and appears to improve theexpansion rate, perhaps because of an improvement in the circulation ofblood or other nutrients through tissue. There is no specific maximumcycle rate, but approximately 100 times per minute is a useful practicallimit for many tissues in order to avoid the dangers associated withrapid movement and the possibility of tissue tears or ruptures. Moretypical cycle rates are from 1 cycle per 10 minutes to 50 cycles perminute, preferably from 1 to 25 cycles per minute, and more preferablyfrom 5 to 15 cycles per minute.

[0082] It is not necessary that the stretch and relax phases have thesame length, nor need they occur continuously. For example, a series ofrelatively rapid stretch and relax cycles (an “active” stage) can befollowed by a relatively long relax phase (an “off” stage) to allowtissue recovery and growth. A typical example of this technique(illustrated with an operating cycle frequency of 10 stretch and relaxcycles per minute) would have an active stage lasting 18 seconds (3cycles) followed by an off stage lasting 42 seconds (7 “cycles”).

[0083] The duration of the tissue expansion process will vary with thehealth and condition of the donor tissue, the extent of expansionnecessary for the particular donor tissue being expanded, the health ofthe patient/recipient of the tissue, and on many other medical factorsthat are outside the scope of this invention. One day (24 hours) can beconsidered to be the lower limit of the time during which tissueexpansion with pulsatile motion is likely to be practiced, from apractical point of view. Shorter periods of time do not providesufficient expansion to require the greater complexity of the invention,relative to standard techniques of tissue expansion. There is also nolimit on maximum duration, other than patient discomfort in in vivosituations and tissue viability during tissue culture. The advantages ofthe method of the invention relative to non-pulsatile techniques will bemore apparent as the duration of treatment increases, so that minimumexpansions of two days or more are preferred.

[0084] These examples of cycles, including factors such as force/timerelationships, relative forces, duration of the process, and the like,should not be considered to be limiting, as many other force/timewaveforms will give satisfactory results of increased expansion raterelative to that which is obtained in the absence of alternatingstretch/relax cycles.

[0085] A typical expanded tissue has an area or volume after beingsubjected to stretching forces (over an appropriate length of time) thatis at least twice that of the tissue prior to being subjected to thestretching forces. The stretched tissue can be used as a graft or (in invitro situations) can be divided into further segments so that one ormore of the further segments is subjected again to the method of theinvention. As long as cell growth continues, new normal tissue can beproduced from parts of the original donor graft. For example, skinhaving a surface area at least two times that of the original donatedskin segment can be provided in one stretching operation (which may lastover several days), usually at least four times, and often at leasteight that of the original. Since the stretched tissue can be dividedand the resulting segments re-stretched, exponential production of skinand other tissue is possible, particularly in in vitro situations.

[0086] Once a stretched tissue graft product has been prepared, it isused in grafting in the same manner as an unstretched graft donortissue.

[0087] The invention now being generally described, the same will bebetter understood by reference to the following detailed examples of theinvention, which are provided here for illustration only and are not tobe considered limiting of the invention unless so specified.

EXAMPLE

[0088] Human full thickness skin was obtained from discarded surgicalmaterial following abdominoplasty under informed consent. Immediatelyupon removal the tissue was placed in 2 liters of sterile transportmedia, epidermal side up and transported to the laboratory at 4° C. Thetissue was defatted using standard aseptic technique, and 6.74 cm²punches were made using a hardened steel blade. The circular tissue wasplaced epidermal side up in the expansion device described in theFigures and in the detailed description above and was attached to thedevice using nylon “T” tags that were inserted through the tissue in aradial pattern of equal spacing. Medium was added, and initial tensionwas set to 100 g per attachment. The device was sealed and the motoractivated to begin force cycling at 10 cycles per minute. The tissuearea was measured prior to initiating cycling. Control tissue was placedin an organ culture device in which the tissue sits on a stainless steelgrid (under no load) or was set up in an expansion device as describedunder tension but was not cycled. The tissue was incubated at 37° C. Themedia was composed of DMEM containing 4.5 g/L glucose and the followingadditives: L-glutamine, 2 mM; hydrocortisone, 0.28 μM; insulin, 4.4 μM;ascorbic acid, 0.3 mM; penicillin, 105 U/L; streptomycin, 100 mg/L. Thedevice was operated in an air atmosphere with CO₂ added for bufferingcapacity and O₂ to prevent hypoxia, as necessary.

[0089] After stretching, the skin was measured and removed from thedevice. The tissue was then prepared for histology, biochemicalanalysis, and animal transplantation. Tissue appeared to be viable underhistological examination and other forms of examination afterstretching.

[0090] After five days in culture full thickness human skin that hadbeen stretched by applying a cycling load increased 3 fold in surfacearea. The tissue remained viable with an intact epidermis and dermis.When skin from the same patient was either placed under no load orplaced under 100 g/attachment of load but not cycling, there was nosignificant increase in surface area that could be measured during thesame time period. This example illustrates that pulsatile tissueexpansion not only allowed rapid tissue expansion, but also allowedtissue expansion to occur at a force less than that necessary to causeexpansion in the absence of pulsatile motion. % Increase in Surface AreaDay 0 1 2 3 4 5 No Load 0 0  0  0 — — Static Load 0 3  1 0  0  0Cyclical Load 0 21  53 120 151 203

[0091] All publications and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

[0092] The invention now being fully described, it will be apparent toone of ordinary skill in the art that many changes and modifications canbe made thereto without departing from the spirit or scope of theappended claims.

What we claim is:
 1. A method of increasing the rate of expansion oftissue area and volume for a given expansion force, either in vivo or invitro, which comprises: preparing a tissue for expansion, and subjectingsaid tissue to stretching forces that alternatively increase anddecrease over multiple cycles during a period of tissue expansion. 2.The method of claim 1, wherein said stretching forces are reduced duringa relax phase to less than 0.9 times the maximum stretching forceapplied during a stretch phase.
 3. The method of claim 1, wherein therelax phase has a minimum stretching force of zero.
 4. The method ofclaim 1, wherein said tissue is an elongated tissue selected from thegroup consisting of tendon, ligament, blood vessel, nerve, ureter, andbowel tissue.
 5. The method of claim 4, wherein said stretching forcesstretch said tissue primarily in one dimension.
 6. The method of claim1, wherein said tissue comprises a skin or mucosal segment.
 7. Themethod of claim 6, wherein said stretching forces stretch said tissueprimarily in two dimensions.
 8. The method of claim 6, wherein said skinsegment is mammalian skin.
 9. The method of claim 8, wherein said skinsegment is human skin.